We propose to develop the capabilities of x-ray spectromicroscopy to allow it to be applied to biochemical imaging of normal and abnormal sperm. This will be done by focusing "soft" X rays (in this case, x rays with a photon energy of 200-800 eV) to the smallest far-field focus of electromagnetic radiation of any wavelength, and scanning a dry or frozen hydrated specimen through that focal spot to form an image. Images at a series of photon energies will then be combined to deliver chemical-state-sensitive x-ray absorption spectra from an entire sperm at better than 50 nm spatial resolution. From this data, major biochemical constituents in individual sperm will be determined by fitting to reference spectra of isolated compounds; furthermore, expected and possibly unexpected spatial correlations between components will be studied using principal component analysis methods. This work will be carried out using x-ray microscopes developed by our group at Stony Brook which operate at a soft x-ray undulator beamline at the National Synchrotron Light Source at Brookhaven National Laboratory. The microscopes will be improved by the addition of better order sorting optics for quatitative spectroscopy, and by equipping a cryo microscope with a more accurate piezo stage and a higher efficiency detector for studies of frozen hydrated specimens without excessive radiation damage. These instrumentation developments will be guided by our goal of studying the correlation between morphological and biochemical variations in sperm, in order to better understand the causes of male infertility. By obtaining x-ray spectromicroscopic data on different sperm morphologies in infertile males, we hope to guide the in vitro fertilization (IVP) clinician in the choice of sperm use for intracytoplasmic sperm injection (ICSI) so as to improve the success rate of the procedure. Spectromicroscopic data anaysis software will be made available for free downloading by other researchers as it is developed.